Albumin-proaerolysin prodrugs

ABSTRACT

The present invention relates to the field of cancer. More specifically, the present invention provides compositions and methods for treating cancer using albumin-proaerolysin prodrugs. Accordingly, in one aspect, the present invention provides prodrug compositions. In certain embodiments, a prodrug composition comprises a prostate-specific antigen (PSA)-activated pro-aerolysin (PA), wherein a PSA cleavable linker replaces the native furin cleavage site within PA; and human serum albumin (HSA) or a fragment thereof fused to the N-terminus of the PSA-activated PA.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. § 371 U.S. national entry ofInternational Application PCT/US2016/013516, having an internationalfiling date of Jan. 15, 2016, which claims the benefit of U.S.Provisional Application No. 62/104,275, filed Jan. 16, 2015, the contentof each of the aforementioned applications is herein incorporated byreference in their entirety.

STATEMENT OF GOVERNMENTAL INTEREST

This invention was made with government support under grant no.CA058236, awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to the field of cancer. More specifically,the present invention provides compositions and methods for treatingcancer using albumin-proaerolysin prodrugs.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

This application contains a sequence listing. It has been submittedelectronically via EFS-Web as an ASCII text file entitled“P12343-02_ST25.txt.” The sequence listing is 61,919 bytes in size, andwas created on Jan. 14, 2016. It is hereby incorporated by reference inits entirety.

BACKGROUND OF THE INVENTION

Following systemic delivery, an effective drug for a metastatic prostatecancer patient must selectively kill malignant cells without producingunacceptable off-target side effects (i.e., unacceptable levels ofkilling or injuring normal cells in host tissue). This is a dauntingengineering challenge. Based upon the fact that cancer cells oftenacquire addiction to specific oncogenic signaling pathways, a newapproach has emerged focused upon designing drugs to selectively inhibitonly particular oncogenic signaling protein targets. In theory, suchhighly selective oncogene-based inhibitors target growth suppressionand/or death of individual oncogene-addicted cancer cells sparing hostnormal cells. As a class, these new oncogene-targeted inhibitors areless toxic than chemotherapeutics, but they are not without sideeffects. More significantly, their therapeutic efficacy is limited byheterogeneity within the cancer cell population with regards toaddiction to the specific oncogenic signaling resulting in drugresistance. Accordingly, new approaches are necessary to overcome tumorcell heterogeneity based therapeutic resistance.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the development ofa fusion protein in which albumin is fused to a variant proaerolysinprodrug. In a specific embodiment of the prodrug composition, theC-terminus of Human Serum albumin (HSA) is fused via a Prostate SpecificAntigen (PSA) cleavable peptide linker (e.g., HSSKLQ, SEQ ID NO: 2) tothe N-terminus of PSA-proaerolysin to generate a novel recombinantfusion protein which will not bind to GPI-anchored proteins on normalcells in the blood or host tissues. Instead, it will accumulate via anenhanced permeability and retention (EPR) effect within sites ofmetastatic prostate cancer where enzymatically active PSA in theextracellular fluid will hydrolyze the HSA linker liberatingPSA-proaerolysin. Additionally, PSA will also remove the C-terminalinhibitory peptide from PSA-proaerolysin to generate aerolysin monomerswhich can oligomerize to form the heptameric pores that will result inselective killing of cells only in the site of metastatic prostatecancer.

Accordingly, in one aspect, the present invention provides prodrugcompositions. In certain embodiments, a prodrug composition comprises aprostate-specific antigen (PSA)-activated pro-aerolysin (PA), wherein aPSA cleavable linker replaces the native furin cleavage site within PA;and human serum albumin (HSA) or a fragment thereof fused to theN-terminus of the PSA-activated PA. In one embodiment, the PSA cleavablelinker comprises SEQ ID NO:5. In another embodiment, the PSA cleavablelinker replaces the amino acids at position 427-432 of SEQ ID NO:2. Inyet another embodiment, the HSA or fragment thereof comprises theC-terminal end of HSA. In a specific embodiment, the HSA or fragmentthereof comprises SEQ ID NO:27.

In yet another specific embodiment, the HSA or fragment thereof is fusedto the N-terminus of the PSA-activated PA with at least onePSA-cleavable linker sequence. In a particular embodiment, the at leastone PSA-cleavable linker sequence comprises SEQ ID NO:5. The at leastone PSA-cleavable linker sequence can be a series of identical linkersequences or a combination of sequences and includes at least 1, 2, 3,4, 5, 6, 7, 8, 9, 10 or more linker sequences. In a more particularembodiment, the HSA or fragment thereof is fused to the N-terminus ofthe PSA-activated PA with four identical PSA-cleavable linker sequences,wherein the linker sequence comprises SEQ ID NO:5.

The present invention also provides a recombinant protein comprising SEQID NO:48. In a specific embodiment, the protein further comprises apolyhistidine tag. In a more specific embodiment, the polyhistidine tagcomprises six histidines at the C-terminus of SEQ ID NO:48.

The present invention also provides a prodrug composition comprising aprostate-specific protease-activated pro-aerolysin (PA), wherein aprostate-specific protease cleavable linker replaces the native furincleavage site within PA; and a blood plasma protein or a fragmentthereof fused to the N-terminus of the PSA-activated PA. In oneembodiment, the prostate-specific protease comprises PSA, prostatespecific membrane antigen (PSMA), or human glandular kallikrein 2 (HK2).In certain embodiments, the blood plasma protein comprises albumin. In amore specific embodiment, the blood plasma protein comprises human serumalbumin.

DETAILED DESCRIPTION OF THE INVENTION

It is understood that the present invention is not limited to theparticular methods and components, etc., described herein, as these mayvary. It is also to be understood that the terminology used herein isused for the purpose of describing particular embodiments only, and isnot intended to limit the scope of the present invention. It must benoted that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include the plural reference unless the contextclearly dictates otherwise. Thus, for example, a reference to a“protein” is a reference to one or more proteins, and includesequivalents thereof known to those skilled in the art and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Specific methods, devices, andmaterials are described, although any methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the present invention.

All publications cited herein are hereby incorporated by referenceincluding all journal articles, books, manuals, published patentapplications, and issued patents. In addition, the meaning of certainterms and phrases employed in the specification, examples, and appendedclaims are provided. The definitions are not meant to be limiting innature and serve to provide a clearer understanding of certain aspectsof the present invention.

I. Definitions

Aerolysin: A channel-forming toxin produced as an inactive protoxincalled proaerolysin (PA) (wild-type PA is shown in SEQ ID NOS: 1 and 2).The PA protein contains many discrete functionalities that include abinding domain (approximately amino acids 1-83 of SEQ ID NO: 2), a toxindomain (approximately amino acids 84-426 of SEQ ID NO: 2), and aC-terminal inhibitory peptide domain (approximately amino acids 427-470of SEQ ID NO: 2) that contains a protease activation site (amino acids427-432 of SEQ ID NO: 2).

The binding domain recognizes and binds to glycophosphatidylinositol(GPI) membrane anchors including those found in Thy-1 on T lymphocytes,the PIGA gene product found in erythrocyte membranes, and Prostate StemCell Antigen (PSCA). Most mammalian cells express GPI anchored proteinson their surfaces. The activation or proteolysis site within wildtype PAis a six amino acid sequence that is recognized as a proteolyticsubstrate by the furin family of proteases. Wild-type PA is activatedupon hydrolysis of a C-terminal inhibitory segment by furin. Activatedaerolysin binds to GPI-anchored proteins in the cell membrane and formsa heptamer that inserts into the membrane producing well-definedchannels of ˜17 Å. Channel formation leads to rapid cell death vianecrosis. Wild-type aerolysin is toxic to mammalian cells, includingerythrocytes, for example at 1 nanomolar or less.

Antibody: Immunoglobulin molecules and immunologically active portionsof immunoglobulin molecules, i.e., molecules that contain an antigenbinding site which specifically binds (immunoreacts with) an antigen. Anaturally occurring antibody (e.g., IgG) includes four polypeptidechains, two heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds. However, the antigen-binding function of an antibodycan be performed by fragments of a naturally occurring antibody. Thus,these antigen-binding fragments are also intended to be designated bythe term antibody. Examples of binding fragments encompassed within theterm antibody include (i) an Fab fragment consisting of the VL, VH, CLand CHI domains; (ii) an Fd fragment consisting of the VH and CHIdomains; (iii) an Fv fragment consisting of the VL and VH domains of asingle arm of an antibody, (iv) a dAb fragment which consists of a VHdomain; (v) an isolated complimentarily determining region (CDR); and(vi) an F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region. Furthermore,although the two domains of the Fv fragment are coded for by separategenes, a synthetic linker can be made that enables them to be made as asingle protein chain (known as single chain Fv (scFv)) by recombinantmethods. Such single chain antibodies are also included. In oneembodiment, an antibody includes camelized antibodies.

In one example, antibody fragments are capable of crosslinking theirtarget antigen, e.g., bivalent fragments such as F(ab′)₂ fragments.Alternatively, an antibody fragment which does not itself crosslink itstarget antigen (e.g., a Fab fragment) can be used in conjunction with asecondary antibody which serves to crosslink the antibody fragment,thereby crosslinking the target antigen. Antibodies can be fragmentedusing conventional techniques and the fragments screened for utility inthe same manner as described for whole antibodies. An antibody isfurther intended to include bispecific and chimeric molecules thatspecifically bind the target antigen.

Specifically binds: Binding that occurs between such paired species asenzyme/substrate, receptor/agonist, antibody/antigen, andlectin/carbohydrate which may be mediated by covalent or non-covalentinteractions or a combination of covalent and non-covalent interactions.When the interaction of the two species produces a non-covalently boundcomplex, the binding which occurs is typically electrostatic,hydrogen-bonding, or the result of lipophilic interactions. Accordingly,“specific binding” occurs between a paired species where there isinteraction between the two which produces a bound complex having thecharacteristics of an antibody/antigen or enzyme/substrate interaction.In particular, the specific binding is characterized by the binding ofone member of a pair to a particular species and to no other specieswithin the family of compounds to which the corresponding member of thebinding member belongs. Thus, for example, an antibody typically bindsto a single epitope and to no other epitope within the family ofproteins. In some embodiments, specific binding between an antigen andan antibody will have a binding affinity of at least 10⁻⁶ M. In otherembodiments, the antigen and antibody will bind with affinities of atleast 10⁻⁷ M, 10⁻⁸ M to 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, or 10⁻¹² M. As usedherein, the terms “specific binding” or “specifically binding” when usedin reference to the interaction of an antibody and a protein or peptidemeans that the interaction is dependent upon the presence of aparticular structure (i.e., the epitope) on the protein.

Cancer: Malignant neoplasm that has undergone characteristic anaplasiawith loss of differentiation, increase rate of growth, invasion ofsurrounding tissue, and is capable of metastasis.

cDNA (complementary DNA): A piece of DNA lacking internal, non-codingsegments (introns) and regulatory sequences which determinetranscription. cDNA can be synthesized in the laboratory by reversetranscription from messenger RNA extracted from cells.

Chemical synthesis: An artificial means by which one can make a proteinor peptide. A synthetic protein or peptide is one made by suchartificial means.

Chemotherapy: In cancer treatment, chemotherapy refers to theadministration of one or a combination of compounds to kill or slow thereproduction of rapidly multiplying cells. Chemotherapeutic agentsinclude those known by those skilled in the art, including, but notlimited to: 5-fluorouracil (5-FU), azathioprine, cyclophosphamide,antimetabolites (such as Fludarabine), antineoplastics (such asEtoposide, Doxorubicin, methotrexate, and Vincristine), carboplatin,cis-platinum and the taxanes, such as taxol and taxotere. Such agentscan be co-administered with the disclosed variant PA fusion proteins toa subject. Alternatively or in addition, chemotherapeutic agents can beadministered prior to and/or subsequent to administration of thedisclosed variant PA fusion proteins to a subject. In one example,chemotherapeutic agents are co-administered with hormonal and radiationtherapy, along with the disclosed variant PA fusion proteins, fortreatment of a localized prostate carcinoma.

Conservative substitution: One or more amino acid substitutions (forexample 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more residues) for amino acidresidues having similar biochemical properties. Typically, conservativesubstitutions have little to no impact on the activity of a resultingpolypeptide. For example, ideally, a modified PA peptide including oneor more conservative substitutions retains proaerolysin activity. Apolypeptide can be produced to contain one or more conservativesubstitutions by manipulating the nucleotide sequence that encodes thatpolypeptide using, for example, standard procedures such assite-directed mutagenesis or PCR.

Substitutional variants are those in which at least one residue in theamino acid sequence has been removed and a different residue inserted inits place. Examples of amino acids which may be substituted for anoriginal amino acid in a protein and which are regarded as conservativesubstitutions include: Ser for Ala; Lys for Arg; Gln or His for Asn; Glufor Asp; Ser for Cys; Asn for Gln; Asp for Glu; Pro for Gly; Asn or Glnfor His; Leu or Val for Ile; Ile or Val for Leu; Arg or Gln for Lys; Leuor Ile for Met; Met, Leu or Tyr for Phe; Thr for Ser; Ser for Thr; Tyrfor Trp; Trp or Phe for Tyr; and Ile or Leu for Val.

Permissive substitutions are non-conservative amino acid substitutions,but also do not significantly alter proaerolysin activity. An example issubstitution of Cys for Ala at position 300 of SEQ ID NO: 2 or 4.Further information about conservative substitutions can be found in,among other locations in, Ben-Bassat et al., (J. Bacteria 169:751-7,1987), O'Regan et al., (Gene 77:237-51, 1989), Sahin-Toth et al.,(Protein Sci. 3:240-7, 1994), Hochuli et al., (Bio/Technology 6:1321-5,1988), WO 00/67796 (Curd et al.) and in standard textbooks of geneticsand molecular biology. In one example, such variants can be readilyselected for additional testing by performing an assay to determine ifthe variant retains variant PA fusion protein activity.

Deletion: The removal of a sequence of a nucleic acid, for example DNA,the regions on either side being joined together.

DNA: Deoxyribonucleic acid. DNA is a long chain polymer which comprisesthe genetic material of most living organisms (some viruses have genescomprising ribonucleic acid, RNA). The repeating units in DNA polymersare four different nucleotides, each of which comprises one of the fourbases, adenine, guanine, cytosine and thymine bound to a deoxyribosesugar to which a phosphate group is attached. Triplets of nucleotides,referred to as codons, in DNA molecules code for amino acid in apolypeptide. The term codon is also used for the corresponding (andcomplementary) sequences of three nucleotides in the mRNA into which theDNA sequence is transcribed.

Enhance: To improve the quality, amount, or strength of something. Inone embodiment, a therapy enhances the ability of a subject to reducetumors, such as a prostate carcinoma, in the subject if the subject ismore effective at fighting tumors. In another embodiment, a therapyenhances the ability of an agent to reduce tumors, such as a prostatecarcinoma, in a subject if the agent is more effective at reducingtumors. Such enhancement can be measured using the methods disclosedherein, for example determining the decrease in tumor volume.

Functional Deletion: A mutation, partial or complete deletion,insertion, or other variation made to a gene sequence which renders thatpart of the gene sequence non-functional. For example, functionaldeletion of a PA binding domain results in a decrease in the ability ofPA to bind to and concentrate in the cell membrane. This functionaldeletion can be reversed by inserting another functional binding domaininto proaerolysin, such as a prostate-specific binding domain, forexample, an LHRH peptide.

Examples of methods that can be used to functionally delete aproaerolysin binding domain, include, but are not limited to: deletionof about amino acids 1-83 of SEQ ID NO: 2 or fragments thereof, such asabout amino acids 45-66 of SEQ ID NO: 2, or inserting one or more of thefollowing mutations into a variant proaerolysin sequence W45A, 147E,M57A, Y61A, K66Q (amino acid numbers refer to SEQ ID NO: 2) (forexample, see Mackenzie et al. J. Biol. Chem. 274: 22604-22609, 1999). Inanother example, functional deletion of a native PA furin cleavage siteresults in a decrease in the ability of PA to be cleaved and activatedby furin, when compared to a wild-type PA molecule.

Immobilized: Bound to a surface, such as a solid surface. A solidsurface can be polymeric, such as polystyrene or polypropylene. In oneembodiment, the solid surface is in the form of a bead. In anotherembodiment, the surface includes a modified PA toxin, and in someexamples further includes one or more prostate-specific binding ligands,such as LHRH peptide, PSMA antibody, and PSMA single chain antibody.Ideally, the modified PA toxin is liberated from the bead once the beadreaches the prostate cell target. Methods of immobilizing peptides on asolid surface can be found in WO 94/29436, and U.S. Pat. No. 5,858,358.Examples of how the molecules can be attached to the bead include, butare not limited to: HSA-PSA cleavage site/linker-PA-bead-prostatebinding ligand; or prostate binding ligand-bead-HSA-cleavage linker-PA.

Isolated: An “isolated” biological component (such as a nucleic acidmolecule or protein) has been substantially separated or purified awayfrom other biological components in the cell of the organism in whichthe component naturally occurs (i.e., other chromosomal andextrachromosomal DNA and RNA). Nucleic acids and proteins that have been“isolated” include nucleic acids and proteins purified by standardpurification methods. The term also embraces nucleic acids and proteinsprepared by recombinant expression in a host cell as well as chemicallysynthesized nucleic acids and proteins. An isolated cell is one whichhas been substantially separated or purified away from other biologicalcomponents of the organism in which the cell naturally occurs.

Malignant: Cells that have the properties of anaplasia invasion andmetastasis.

Mammal: This term includes both human and non-human mammals. Similarly,the terms “subject” and “patient” are interchangeable and include bothhuman and veterinary subjects. Examples of mammals include, but are notlimited to, humans, pigs, cows, goats, cats, dogs, rabbits and mice.

Neoplasm: Abnormal growth of cells.

Normal Cell: Non-tumor cell, non-malignant, uninfected cell.

Oligonucleotide: A linear polynucleotide sequence of up to about 200nucleotide bases in length, for example a polynucleotide (such as DNA orRNA) which is at least about 6 nucleotides, for example at least 15, 50,100 or 200 nucleotides long.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein coding regions, in the samereading frame.

ORF (open reading frame): A series of nucleotide triplets (codons)coding for amino acids without any termination codons. These sequencesare usually translatable into a peptide.

Polynucleotide: A linear nucleic acid sequence of any length. Therefore,a polynucleotide includes molecules which are at least 5, 15, 50, 100,200, 400, 500, 1000, 1100, or 1200 (oligonucleotides) and alsonucleotides as long as a full-length cDNA or chromosome.

Proaerolysin: The inactive protoxin of aerolysin. The cDNA and proteinof a wild-type or native proaerolysin (PA) are shown in SEQ ID NOS: 1and 2, respectively. In one example, a variant or modified proaerolysinmolecule includes a prostate-specific protease cleavage site, such as aPSA-specific cleavage site, which permits activation of the variant PAin the presence of a prostate-specific protease such as PSA, PMSA, orHK2. In one example, a prostate-specific protease cleavage site isinserted into the native furin cleavage site of PA, such that PA isactivated in the presence of a prostate-specific protease, but notfurin. Alternatively, the furin cleavage site can be functionallydeleted using mutagenesis of the six amino acid sequence, and aprostate-specific protease cleavage sequence can be inserted. In anotherexample, a variant PA molecule further includes deletion or substitutionof one or more of the native PA amino acids. In yet another example, avariant PA molecule further includes another molecule (such as anantibody or peptide) linked or added to (or within) the variant PAmolecule. In another example, a variant PA molecule includes aprostate-tissue specific binding domain.

In another example, a variant PA molecule further includes afunctionally deleted binding domain (about amino acids 1-83 of SEQ IDNO: 2). Functional deletions can be made using any method known in theart, such as deletions, insertions, mutations, or substitutions.Examples include, but are not limited to deleting the entire bindingdomain (or portions thereof) or introduction of point mutations, whichresult in a binding domain with decreased function. For example, a PAmolecule which has a functionally deleted binding domain (and no bindingsequence substituted therefor), will have a decreased ability toaccumulate in a cell membrane, and therefore lyse cells at a slower ratethan a wild-type PA sequence. Also disclosed are variant PA proteins inwhich the native binding domain is functionally deleted and replacedwith a prostate-tissue specific binding domain as described below.

In another example, a variant or modified PA molecule includes a PSAcleavage site, and a functionally deleted binding domain which isreplaced with a prostate-tissue specific binding domain. Such variant PAfusion proteins are targeted to prostate cells via the prostate-tissuespecific binding domain, and activated in the presence of PSA.

Particular non-limiting examples of variant PSA proteins are shown inSEQ ID NOS: 4, 7, 10, 13, 24, and 25.

Modified PA activity is the activity of an agent in which the lysis ofcells is affected. Cells include, but are not limited toprostate-specific protease secreting cells, such as PSA-secreting cells,such as prostate cancer cells, such as slow-proliferating prostatecancer cells. Agents include, but are not limited to, modified PAproteins, nucleic acids, specific binding agents, including variants,mutants, polymorphisms, fusions, and fragments thereof, disclosedherein. In one example, modified PA activity is said to be enhanced whenmodified PA proteins or nucleic acids, when contacted with aPSA-secreting cell (such as a prostate cancer cell), promote lysis anddeath of the cell, for example by at least 10%, or for example by atleast 25%, 50%, 100%, 200% or even 500%, when compared to lysis of anon-PSA producing cell. In other examples, modified PA activity is saidto be enhanced when modified PA proteins and nucleic acids, whencontacted with a tumor, decrease tumor cell volume, such as a prostatetumor, for example by at least 10% for example by at least 20%, 30%,40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or even 100% (completeelimination of the tumor). Assays which can be used to determine if anagent has modified PA activity are described, for example, in U.S. Pat.Nos. 7,838,266, 7,745,395, and 7,282,476, which are all incorporatedherein by reference.

Promoter: An array of nucleic acid control sequences which directtranscription of a nucleic acid. A promoter includes necessary nucleicacid sequences near the start site of transcription, such as, in thecase of a polymerase II type promoter, a TATA element. A promoter alsooptionally includes distal enhancer or repressor elements which can belocated as much as several thousand base pairs from the start site oftranscription.

Prostate-specific promoter: A promoter responsive to testosterone andother androgens, which therefore promotes gene expression in prostatecells. Examples include, but are not limited to the probasin promoter;the prostate specific antigen (PSA) promoter; the prostate specificmembrane antigen (PSMA) promoter; and the human glandular kallikrein 2(HK2) promoter.

Prostate-specific protease cleavage site: A sequence of amino acidswhich is recognized and specifically and efficiently hydrolyzed(cleaved) by a prostate-specific protease. Examples include, but are notlimited to a PSA-specific cleavage site, a PSMA-specific cleavage siteand an HK2-specific cleavage site. Variant PA fusion proteins of thepresent invention can comprise one or more cleavage sites/linkers. Forexample, albumin can be fused to the N-terminus of a variant PA proteinusing one, two, three, four, five, six or more prostate-specificprotease cleavage site linkers.

PSA-specific cleavage site: is a sequence of amino acids which isrecognized and specifically and efficiently hydrolyzed (cleaved) byprostate specific antigen (PSA). Such peptide sequences can beintroduced into other molecules, such as PA, to produce prodrugs thatare activated by PSA. Upon activation of the modified PA by PSA, PA isactivated and can exert its cytotoxicity. Examples of PSA-specificcleavage sites include, but are not limited to, those shown in SEQ IDNOS: 5, 8 and 14-21, those disclosed in U.S. Pat. Nos. 6,391,305;6,368,598; 6,265,540; 5,998,362; 5,948,750; and 5,866,679.

PSMA-specific cleavage site: Particular examples of PSMA-specificcleavage sites can be found in WO/0243773 to Isaacs and Denmeade (hereinincorporated by reference). The PSMA cleavage site includes at least thedipeptide, X₁X₂. This peptide contains the amino acids Glu or Asp atposition X₁. X₂ can be Glu, Asp, Gln, or Asn. Tripeptides X₁X₂X₃ arealso suitable, with X₁ and X₂ defined as before, with X₃ as Glu, Asp,Gln or Asn. Tetrapeptides X₁X₂X₃X₄ are also suitable, with X₁₋₃ definedas above, and with X₄ as Glu, Asp, Gln or Asn. Pentapeptides X₁X₂X₃X₄X₅are also suitable, with X₁₋₄ defined as above, and with X₅ as Glu, Asp,Gln or Asn. Hexapeptides X₁X₂X₃X₄X₅X₆ are also suitable, with X₁₋₅defined as above, and with X₆ as Glu, Asp, Gln or Asn. Further peptidesof longer sequence length can be constructed in similar fashion.

Generally, the peptides are of the following sequence: X₁ . . . X_(n),where n is 2 to 30, preferably 2 to 20, more preferably 2 to 15, andeven more preferably 2 to 6, where X₁ is Glu, Asp, Gln or Asn, but ispreferably Glu or Asp, and X₂-X_(n) are independently selected from Glu,Asp, Gln and Asn. Some preferred peptide sequences are as above, exceptthat X₂-X_(n-1) are independently selected from Glu, and Asp, and X_(n)is independently selected from Glu, Asp, Gln and Asn. The length of thepeptide can be optimized to allow for efficient PSMA hydrolysis,enhanced solubility of therapeutic drug in aqueous solution, if this isneeded, and limited non-specific cytotoxicity in vitro.

HK2-specific cleavage site: Particular examples of HK2-specific cleavagesites are disclosed in WO01/09165 and U.S. Patent Publication No.20120309692 and include, but are not limited to, Lys-Arg-Arg,Ser-Arg-Arg, Ala-Arg-Arg, His-Arg-Arg, Gln-Arg-Arg, Ala-Phe-Arg,Ala-Gln-Arg, Ala-Lys-Arg, Ala-Arg-Lys, Ala-His-Arg, Gln-Lys-Arg-Arg (SEQID NO:28), Lys-Ser-Arg-Arg (SEQ ID NO:29), Ala-Lys-Arg-Arg (SEQ IDNO:30), Lys-Lys-Arg-Arg (SEQ ID NO:31), His-Lys-Arg-Arg (SEQ ID NO:32),Lys-Ala-Phe-Arg (SEQ ID NO:33), Lys-Ala-Gln-Arg (SEQ ID NO:34),Lys-Ala-Lys-Arg (SEQ ID NO:35), Lys-Ala-Arg-Lys (SEQ ID NO:36),Lys-Ala-His-Arg (SEQ ID NO:37), His-Ala-Gln-Lys-Arg-Arg (SEQ ID NO:38),Gly-Gly-Lys-Ser-Arg-Arg (SEQ ID NO:39), His-Glu-Gln-Lys-Arg-Arg (SEQ IDNO:40), His-Glu-Ala-Lys-Arg-Arg (SEQ ID NO:41), Gly-Gly-Gln-Lys-Arg-Arg(SEQ ID NO:42), His-Glu-Gln-Lys-Arg-Arg (SEQ ID NO:43),Gly-Gly-Ala-Lys-Arg-Arg (SEQ ID NO:44), His-Glu-Gln-Lys-Arg-Arg (SEQ IDNO:45), Gly-Gly-Lys-Lys-Arg-Arg (SEQ ID NO:46), andGly-Gly-His-Lys-Arg-Arg (SEQ ID NO:47).

PRX302: A modified proaerolysin where the furin site of proaerolysin hasbeen replaced with a PSA-specific cleavage site. SEQ ID NOS: 3 and 4show the PRX302 cDNA and protein sequence, respectively. SEQ ID NO:26shows the protein sequence of SEQ ID NO: 4 with an N-terminal His tag.The term “PRX302” includes the proteins of both SEQ ID NO: 4 and SEQ IDNO:26.

Prostate tissue-specific binding domain: A molecule, such as a peptideligand, toxin, or antibody, which has a higher specificity for prostatecells than for other cell types. In one example, a prostate tissuespecific binding domain has a lower K_(D) in prostate tissue or cellsthan in other cell types, (i.e., binds selectively to prostate tissuesas compared to other normal tissues of the subject), for example atleast a 10-fold lower K_(D), such as an at least 20-, 50-, 75-, 100- oreven 200-fold lower K_(D). Such sequences can be used to target anagent, such as a variant PA molecule, to the prostate. Examples include,but are not limited to: antibodies which recognize proteins that arerelatively prostate-specific such as PSA, PSMA, HK2, prostasin, andhepsin; ligands which have prostate-selective receptors such as naturaland synthetic luteinizing hormone releasing hormone (LHRH); andendothelin (binding to cognate endothelin receptor).

Purified: The term “purified” does not require absolute purity; rather,it is intended as a relative term. Thus, for example, a substantiallypurified protein or nucleic acid preparation (such as the modified PAtoxins disclosed herein) is one in which the protein or nucleic acidreferred to is more pure than the protein in its natural environmentwithin a cell or within a production reaction chamber (as appropriate).For example, a preparation of a modified PA protein is purified if theprotein represents at least 50%, for example at least 70%, of the totalprotein content of the preparation. Methods for purification of proteinsand nucleic acids are well known in the art. Examples of methods thatcan be used to purify a protein, such as a modified PA, include, but arenot limited to the methods disclosed in Sambrook et al. (MolecularCloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989, Ch. 17).

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination is often accomplished by chemical synthesisor, more commonly, by the artificial manipulation of isolated segmentsof nucleic acids, e.g., by genetic engineering techniques. A recombinantprotein is one that results from expressing a recombinant nucleic acidencoding the protein.

Sample: Biological samples containing genomic DNA, cDNA, RNA, or proteinobtained from the cells of a subject, such as those present inperipheral blood, urine, saliva, semen, tissue biopsy, surgicalspecimen, fine needle aspirates, amniocentesis samples and autopsymaterial. In one example, a sample includes prostate cancer cellsobtained from a subject.

Sequence identity/similarity: The identity/similarity between two ormore nucleic acid sequences, or two or more amino acid sequences, isexpressed in terms of the identity or similarity between the sequences.Sequence identity can be measured in terms of percentage identity; thehigher the percentage, the more identical the sequences are. Sequencesimilarity can be measured in terms of percentage similarity (whichtakes into account conservative amino acid substitutions); the higherthe percentage, the more similar the sequences are.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smith &Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol.Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp,CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988;Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; andPearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J.Mol. Biol. 215:403-10, 1990, presents a detailed consideration ofsequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403-10, 1990) is available from several sources,including the National Center for Biological Information (NCBI, NationalLibrary of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894) andon the Internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. Additionalinformation can be found at the NCBI web site.

For comparisons of amino acid sequences of greater than about 30 aminoacids, the Blast 2 sequences function is employed using the defaultBLOSUM62 matrix set to default parameters, (gap existence cost of 11,and a per residue gap cost of 1). When aligning short peptides (fewerthan around 30 amino acids), the alignment should be performed using theBlast 2 sequences function, employing the PAM30 matrix set to defaultparameters (open gap 9, extension gap 1 penalties). Proteins with evengreater similarity to the reference sequence will show increasingpercentage identities when assessed by this method, such as at least70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98 or even 99% sequence identity. When less than the entiresequence is being compared for sequence identity, homologs willtypically possess at least 75% sequence identity over short windows of10-20 amino acids, and can possess sequence identities of at least 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even99% depending on their identity to the reference sequence. Methods fordetermining sequence identity over such short windows are described atthe NCBI web site.

Protein homologs are typically characterized by possession of at least70%, such as at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or even 99% sequence identity, counted overthe full-length alignment with the amino acid sequence using the NCBIBasic Blast 2.0, gapped blastp with databases such as the nr orswissprot database. Queries searched with the blastn program arefiltered with DUST (Hancock and Armstrong, 1994, Comput. Appl. Biosci.10:67-70). Other programs use SEG.

One of skill in the art will appreciate that these sequence identityranges are provided for guidance only; it is possible that stronglysignificant homologs could be obtained that fall outside the rangesprovided. Provided herein are the peptide homologs described above, aswell as nucleic acid molecules that encode such homologs.

Nucleic acid sequences that do not show a high degree of identity maynevertheless encode identical or similar (conserved) amino acidsequences, due to the degeneracy of the genetic code. Changes in anucleic acid sequence can be made using this degeneracy to producemultiple nucleic acid molecules that all encode substantially the sameprotein. Such homologous peptides can, for example, possess at least75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or even 99% sequence identity determined by this method. Whenless than the entire sequence is being compared for sequence identity,homologs can, for example, possess at least 75%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even 99% sequenceidentity over short windows of 10-20 amino acids. Methods fordetermining sequence identity over such short windows can be found atthe NCBI web site. One of skill in the art will appreciate that thesesequence identity ranges are provided for guidance only; it is possiblethat significant homologs or other variants can be obtained that falloutside the ranges provided.

Subject: Living multicellular vertebrate organisms, a category whichincludes both human and veterinary subjects that require an increase inthe desired biological effect. Examples include, but are not limited to:humans, apes, dogs, cats, mice, rats, rabbits, horses, pigs, and cows.The term “subject” can be used interchangeably with the term “patient.”

Therapeutically Effective Amount: An amount sufficient to achieve adesired biological effect, for example, an amount that is effective todecrease the size (i.e., volume), side effects and/or metastasis ofprostate cancer. In one example, it is an amount sufficient to decreasethe symptoms or effects of a prostate carcinoma, such as the size of thetumor. In particular examples, it is an amount effective to decrease thesize of a prostate tumor and/or prostate metastasis by at least 30%,40%, 50%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or even 100% (complete elimination of thetumor).

In particular examples, it is an amount of a variant PA fusion proteineffective to decrease a prostate tumor and/or an amount of prostatecancer cells lysed by a variant PA fusion protein, such as in a subjectto whom it is administered, for example a subject having one or moreprostate carcinomas. In other examples, it is an amount of a variant PAfusion protein and/or an amount of prostate cancer cells lysed by such avariant PA fusion protein, effective to decrease the metastasis of aprostate carcinoma.

In one embodiment, the therapeutically effective amount also includes aquantity of a variant PA fusion protein and/or an amount of prostatecancer cells lysed by a variant PA fusion protein sufficient to achievea desired effect in a subject being treated. For instance, these can bean amount necessary to improve signs and/or symptoms a disease such ascancer, for example prostate cancer.

An effective amount of a variant PA fusion protein and/or prostatecancer cells lysed by such a variant PA fusion protein can beadministered in a single dose, or in several doses, for example daily,during a course of treatment. However, the effective amount of will bedependent on the subject being treated, the severity and type of thecondition being treated, and the manner of administration. For example,a therapeutically effective amount of a variant PA fusion protein canvary from about 1-10 mg per 70 kg body weight, for example about 2.8 mg,if administered iv and about 10-100 mg per 70 kg body weight, forexample about 28 mg, if administered intraprostatically orintratumorally. In addition, a therapeutically effective amount ofprostate cancer cells lysed by PA (variant or wild-type) can vary fromabout 10⁶ to 10⁸ cells.

Therapeutically effective dose: In one example, a dose of a variant PAfusion protein sufficient to decrease tumor cell volume, such as aprostate carcinoma, in a subject to whom it is administered, resultingin a regression of a pathological condition, or which is capable ofrelieving signs or symptoms caused by the condition. In a particularexample, it is a dose of a variant PA fusion protein sufficient todecrease metastasis of a prostate cancer.

In yet another example, it is a dose of cell lysate resulting fromcontact of cells with a variant PA fusion protein sufficient to decreasetumor cell volume, such as a prostate carcinoma, in a subject to whom itis administered, resulting in a regression of a pathological condition,or which is capable of relieving signs or symptoms caused by thecondition. In a particular example, it is a dose of cell lysateresulting from contact of cells with a modified or wild-type PAsufficient to decrease metastasis of a prostate cancer.

Tumor: A neoplasm. Includes solid and hematological (or liquid) tumors.Examples of hematological tumors include, but are not limited to:leukemias, including acute leukemias (such as acute lymphocyticleukemia, acute myelocytic leukemia, acute myelogenous leukemia andmyeloblastic, promyelocytic, myelomonocytic, monocytic anderythroleukemia), chronic leukemias (such as chronic myelocytic(granulocytic) leukemia, chronic myelogenous leukemia, and chroniclymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease,non-Hodgkin's lymphoma (including low-, intermediate-, and high-grade),multiple myeloma, Waldenstrdm's macroglobulinemia, heavy chain disease,myelodysplastic syndrome, mantle cell lymphoma and myelodysplasia.

Examples of solid tumors, such as sarcomas and carcinomas, include, butare not limited to: fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma,mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, coloncarcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lungcancers, ovarian cancer, prostate cancer, hepatocellular carcinoma,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma,renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,Wilms' tumor, cervical cancer, testicular tumor, bladder carcinoma, andCNS tumors (such as a glioma, astrocytoma, medulloblastoma,craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma andretinoblastoma).

Transformed: A transformed cell is a cell into which has been introduceda nucleic acid molecule by molecular biology techniques. As used herein,the term transformation encompasses all techniques by which a nucleicacid molecule might be introduced into such a cell, includingtransfection with viral vectors, transformation with plasmid vectors,and introduction of naked DNA by electroporation, lipofection, andparticle gun acceleration.

Transgenic Cell: Transformed cells which contain foreign, non-nativeDNA.

Transgenic mammal: Transformed mammals which contain foreign, non-nativeDNA. In one embodiment, the non-native DNA is a modified PA whichincludes HSA fused to the N-terminus of PA using a prostate-specificprotease cleavage site.

Variants or fragments or fusion proteins: The production of a variant PAfusion protein can be accomplished in a variety of ways (for example seeExamples 12 and 16 of U.S. Pat. Nos. 7,838,266, 7,745,395, and7,282,476, which are all incorporated herein by reference). DNAsequences which encode for a variant PA fusion protein, or a fragment orvariant of a variant PA fusion protein (for example a fragment orvariant having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or even 99% sequence identity to a variant PA fusionprotein) can be engineered to allow the protein to be expressed ineukaryotic cells or organisms, bacteria, insects, and/or plants. Toobtain expression, the DNA sequence can be altered and operably linkedto other regulatory sequences. The final product, which contains theregulatory sequences and the therapeutic variant PA fusion protein, isreferred to as a vector. This vector can be introduced into eukaryotic,bacteria, insect, and/or plant cells. Once inside the cell the vectorallows the protein to be produced.

A fusion protein which includes a modified PA, (or variants,polymorphisms, mutants, or fragments thereof) linked to other amino acidsequences that do not inhibit the desired activity of the protein, forexample the ability to lyse tumor cells. In one example, the other aminoacid sequences are no more than 5, 6, 7, 8, 9, 10, 20, 30, or 50 aminoacid residues in length. In other embodiments, a modified PA is fused toanother peptide/protein that is more than 50 amino acids in lengthincluding, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198,199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212,213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226,227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240,241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254,255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268,269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282,283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296,297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310,311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324,325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338,339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352,353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366,367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380,381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394,395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408,409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422,423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436,437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450,451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464,465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478,479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492,493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506,507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520,521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534,535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548,549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562,563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576,577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590,591, 592, 593, 594, 595, 596, 597, 598, 599, 600 or more.

One of ordinary skill in the art will appreciate that the DNA can bealtered in numerous ways without affecting the biological activity ofthe encoded protein. For example, PCR can be used to produce variationsin the DNA sequence which encodes a variant PA toxin. Such variants canbe variants optimized for codon preference in a host cell used toexpress the protein, or other sequence changes that facilitateexpression.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector can include nucleic acidsequences that permit it to replicate in the host cell, such as anorigin of replication. A vector can also include one or more selectablemarker genes and other genetic elements known in the art.

II. Variant Proaerolysin Molecules

Bacterial toxins, such as aerolysin produced by Aeromonas hydrophiliaand α-hemolysin produced by Staph aureus, are beta-sheet proteins thatoligomerize in the plasma membrane to produce pores that lead to rapidcytolytic cell death. Pore formation physically disrupts the cellmembranes, and results in death of cells in all phases of the cellcycle, including non-proliferating cells (i.e., G0 arrested). However,wild-type aerolysin kills cells indiscriminately. Herein disclosed is afusion protein comprising human serum albumin and the inactive protoxinform of aerolysin that is activated by cleavage of the activation domainwith a prostate-specific protease that also cleaves the HSA bulk protein(a variant PA) that can be targeted to, and activated by, prostatecancer specific proteins. One advantage of the disclosed variant PAfusion proteins for treatment of localized and metastatic prostatecancer is that it combines a proliferation independent therapy withprostate-specific drug delivery, resulting in minimal side effects topatients. One skilled in the art will understand that other protoxins,such as Clostridium septicum alpha toxin, Bacillus thuringiensisdelta-toxin, and human perforin, bouganin, Pseudomonas exotoxin, Bcl-2,Cholera toxin, Abrin, Ricin, Verotoxin, Diptheria toxin, Tetanus toxin,Botulinum toxin, Neural thread protein, and Ribnuclease A can besubstituted for proaerolysin.

Disclosed herein are variant PA fusion proteins, including both DNA andprotein sequences, which include a prostate-specific protease cleavagesequence. Such variants are also fused with albumin using at least oneprostate-specific protease cleavage sequence/linker (including one, two,three, four, five or more consecutive linkers). Examples ofprostate-specific protease cleavage sequences include, but are notlimited to: PSA, PSMA, and HK2 cleavage sequences. The prostate-specificprotease cleavage sequence functionally replaces the native furincleavage site of wild-type PA. This replacement results in aproaerolysin variant that only becomes cytolytically active in thepresence of enzymatically active proteases such as PSA, PSMA, or HK2.PSA is a serine protease with the ability to recognize and hydrolyzespecific peptide sequences. It is secreted by normal and malignantprostate cells in an enzymatically active form and becomes inactivatedupon entering the circulation. Since neither blood nor normal tissueother than the prostate contains enzymatically active PSA, theproteolytic activity of PSA was used to activate protoxins at sites ofprostate cancer. Any PSA, PSMA, or HK2 cleavage site can be used.Examples of PSA cleavage sites include, but are not limited to, thoseshown in SEQ ID NOS: 5, 8, 11, and 14-21. In a particular example, thePSA cleavage site includes SEQ ID NO: 5.

In some examples, the furin cleavage site of PA (amino acids 427-432 ofSEQ ID NO: 2) is deleted and a prostate-specific protease cleavage site,such as a PSA cleavage site, is inserted. In other examples, the furincleavage site of PA is mutated and a prostate-specific protease cleavagesite, such as a PSA cleavage site, inserted within, or added to the N-or C-terminus of the furin site.

Also disclosed are variant PA fusion proteins in which the PA bindingdomain is functionally deleted. Such variant PA fusion proteins cancontain a native furin cleavage site, whereby targeting to prostatecells is achieved by functionally replacing the PA binding domain with aprostate-tissue specific binding domain. Alternatively, variant PAfusion proteins contain a prostate-specific protease cleavage site,whereby activation of the protoxin primarily occurs in cells thatsecrete a prostate-specific protease. The PA binding domain includesabout amino acids 1-83 of SEQ ID NO: 2. The binding domain can befunctionally deleted using any method known in the art, for example bydeletion of all or some of the amino acids of the binding domain, suchas deletion of amino acids 1-83 of SEQ ID NO: 2 or 4, or such asdeletion of one or more amino acids shown as amino acids 45-66 of SEQ IDNO: 2 or 4. In other examples, the binding domain is functionallydeleted by introduction of one or more site-specific mutations into thevariant PA sequence, such as W45A, 147E, M57A, Y61A, and K66Q of SEQ IDNO: 2 or 4.

Variant PA fusion proteins which include a prostate-tissue specificbinding domain which functionally substitutes for the native PA bindingdomain are disclosed. The use of one or more prostate-tissue specificbinding domains can increase targeting of the disclosed variant PAfusion proteins to the prostate cells and its metastases. Severalprostate-tissue specific binding domains are known. Examples include,but are not limited to a luteinizing hormone releasing hormone (LHRH)sequence, such as those shown in SEQ ID NOS: 22 and 23, and antibodiesthat recognize PSA and/or PSMA.

One or more prostate-tissue specific binding domains can be linked toone or more amino acids of the disclosed variant PA fusion proteins, butideally, do not interfere significantly with the ability of the variantPA to be activated by a prostate-specific protease such as PSA, and theability to form pores in cell membranes. For example, prostate tissuespecific binding domains can be linked or inserted at an N- and/orC-terminus of a variant PA In some examples, the native binding domainof PA is deleted (i.e., amino acids 1-83 of SEQ ID NO: 2 or 4), suchthat attachment or linking of a prostate tissue specific binding domainto the N-terminus results in attachment to amino acid 84 of SEQ ID NO: 2or 4. In other examples, smaller deletions or point mutations areintroduced into the native binding domain of PA, such that attachment orlinking of a prostate tissue specific binding domain to the N-terminusresults in attachment to amino acid 1 of SEQ ID NO: 2 or 4 (or whicheveramino acid is N terminal following functional deletion of the native PAbinding domain). In some examples, the N-terminal amino acid of PA ischanged to a Cys or other amino acid to before attaching aprostate-tissue specific binding domain, to assist in linking theprostate-tissue specific binding domain to the variant PA protein.

Alternatively or in addition, one or more prostate tissue specificbinding domains can be attached or linked to other amino acids of avariant PA molecule, such as amino acid 215 or 300 of SEQ ID NO: 2 or 4.In some examples, a Cys amino acid replaces the native amino acid atthat position. For example, the following changes can be made to SEQ IDNO: 2 or 4: Tyr215Cys or Ala300Cys. In one example, where the prostatetissue specific binding domain is an antibody, crosslinking can be usedto attach antibodies to a variant PA, for example by reacting aminogroups on the antibody with cysteine located in the PA variant (such asamino acids Cys19, Cys75, Cys159, and/or Cys164 of SEQ ID NO: 2).

Also disclosed are particular variant PA fusion proteins, such as thoseshown in SEQ ID NOS: 3, 4, 6, 7, 9, 10, 12, 13, 24 and 25.

In some examples the disclosed variant PA fusion proteins are linked orimmobilized to a surface, such as a bead. The bead can also include aprostate-specific ligand to enhance targeting to a prostate cell, suchas a localized or metastasized prostate cancer cell.

III. Human Serum Albumin

The terms, human serum albumin (HSA) and human albumin (HA) are usedinterchangeably herein. The terms, “albumin and “serum albumin” arebroader, and encompass human serum albumin (and fragments and variantsthereof) as well as albumin from other species (and fragments andvariants thereof).

As used herein, “albumin” refers collectively to albumin protein oramino acid sequence, or an albumin fragment or variant, having one ormore functional activities (e.g., biological activities) of albumin. Inparticular, “albumin” refers to human albumin or fragments thereof (seeEP 201 239, EP 322 094 WO 97/24445, WO95/23857) or albumin from othervertebrates or fragments thereof, or analogs or variants of thesemolecules or fragments thereof.

As used herein, the albumin portion of the fusion protein may comprisethe full length of the sequence as shown in SEQ ID NO:27, or may includeone or more fragments thereof that are capable preventing, substantiallyreducing or reducing binding of the recombinant PRX302 pro-drug proteinto GPI-anchored proteins on normal cells in the blood or host tissues.In one embodiment, the HA protein fragment comprises the N-terminal endof HA. In another embodiment, the HA protein fragment comprises theC-terminal end of HA. In particular embodiments, HA fragments maycomprise 10 or more amino acids in length or may comprise about 15, 20,25, 30, 50, or more contiguous amino acids from the HA sequence or mayinclude part or all of specific domains of HA. For instance, one or morefragments of HA spanning the first two immunoglobulin-like domains maybe used.

The albumin portion of the albumin fusion proteins of the invention maybe a variant of normal HA. The term “variants” includes insertions,deletions and substitutions, either conservative or non-conservative,where such changes do not substantially alter one or more of theoncotic, useful ligand-binding and non-immunogenic properties ofalbumin.

In particular, the albumin fusion proteins of the invention may includenaturally occurring polymorphic variants of human albumin and fragmentsof human albumin, for example those fragments disclosed in EP 322 094(namely HA (Pn), where n is 369 to 419). The albumin may be derived fromany vertebrate, especially any mammal, for example human, cow, sheep, orpig. Non-mammalian albumins include, but are not limited to, hen andsalmon. The albumin portion of the fusion protein may be from adifferent animal than the PRC302 portion.

Generally speaking, an HA fragment or variant will be at least 100 aminoacids long, preferably at least 150 amino acids long. The HA variant mayconsist of or alternatively comprise at least one whole domain of HA,for example domains 1 (amino acids 1-194 of SEQ ID NO:27), 2 (aminoacids 195-387 of SEQ ID NO:27), 3 (amino acids 388-585 of SEQ ID NO:27),1+2 (1-387 of SEQ ID NO:27), 2+3 (195-585 of SEQ ID NO:27 (amino acids1-194 of SEQ ID NO:27+amino acids 388-585 of SEQ ID NO:27). Each domainis itself made up of two homologous subdomains namely 1-105, 120-194,195-291, 316-387, 388-491 and 512-585, with flexible inter-subdomainlinker regions comprising residues Lys106 to Glu119, Glu292 to Val315and Glu492 to Ala511.

In certain embodiments, the albumin portion of an albumin fusion proteinof the invention comprises at least one subdomain or domain of HA orconservative modifications thereof.

The present invention relates generally to fusion proteins comprisingalbumin and methods of treating, preventing, or ameliorating diseases ordisorders. A fusion protein comprising albumin refers to a proteinformed by the fusion of at least one molecule of albumin (or a fragmentor variant thereof) to at least one molecule of a PRX302 protein (orfragment or variant thereof). An albumin-PA or albumin-PRX302 fusionprotein comprises at least a fragment or variant of a PA protein and atleast a fragment or variant of human serum albumin, which are associatedwith one another, preferably by genetic fusion (i.e., the albumin fusionprotein is generated by translation of a nucleic acid in which apolynucleotide encoding all or a portion of a PA/PRX302 protein isjoined in-frame with a polynucleotide encoding all or a portion ofalbumin) or chemical conjugation to one another. The PA/PRX302 proteinand albumin protein, once part of the fusion protein, may be referred toas a “portion”, “region” or “moiety” of the fusion protein.

In one embodiment, the invention provides a fusion protein comprising,or alternatively consisting of, a PA/PRX302 protein and a serum albuminprotein. In other embodiments, the invention provides a fusion proteincomprising, or alternatively consisting of, a biologically active and/ortherapeutically active fragment of a PA/PRX302 protein and a serumalbumin protein. In other embodiments, the invention provides a fusionprotein comprising, or alternatively consisting of, a biologicallyactive and/or therapeutically active variant of a PA/PRX302 protein anda serum albumin protein. In particular embodiments, the serum albuminprotein component of the fusion protein is the mature portion of serumalbumin.

In further embodiments, the invention provides a fusion proteincomprising, or alternatively consisting of, a PA/PRX302 protein, and abiologically active and/or therapeutically active fragment of serumalbumin. In further embodiments, the invention provides a fusion proteincomprising, or alternatively consisting of, a PA/PRX302 protein and abiologically active and/or therapeutically active variant of serumalbumin. In certain embodiments, the PA/PRX302 protein portion of thefusion protein is the full length of the PA/PRX302 protein.

In further embodiments, the invention provides a fusion proteincomprising or alternatively consisting of, a biologically active and/ortherapeutically active fragment or variant of a PA/PRX302 protein and abiologically active and/or therapeutically active fragment or variant ofserum albumin. In some embodiments, the invention provides a fusionprotein comprising, or alternatively consisting of, the mature portionof a PA/PRX302 protein and the mature portion of serum albumin.

In specific embodiments, the fusion protein comprises HA as theN-terminal portion, and a PA/PRX302 protein as the C-terminal portion.Alternatively, a fusion protein comprising HA as the C-terminal portion,and a PA/PRX302 protein as the N-terminal portion may also be used.

In other embodiments, the fusion protein has a PA/PRX302 protein fusedto both the N-terminus and the C-terminus of albumin. In one embodiment,the PA/PRX302 proteins fused at the N- and C-termini are the samePA/PRX302 proteins. In other embodiments, the PA/PRX302 proteins fusedat the N- and C-termini are different PA/PRX302 proteins or justdifferent proteins. In another embodiment, the PA/PRX302 proteins fusedat the N- and C-termini are different therapeutic proteins which may beused to treat or prevent the same disease, disorder, or condition.

In addition to the fusion protein in which the albumin portion is fusedN-terminal and/or C-terminal of the PA/PRX302 protein portion, fusionproteins of the invention may also be produced by inserting thePA/PRX302 protein or peptide of interest into an internal region of HA.For instance, within the protein sequence of the HA molecule a number ofloops or turns exist between the end and beginning of α-helices, whichare stabilized by disulphide bonds. The loops, as determined from thecrystal structure of HA (PDB identifiers 1AO6, 1BJ5, 1BKE, 1BM0, 1E7E to1E7I and 1UOR) for the most part extend away from the body of themolecule. These loops are useful for the insertion, or internal fusion,of therapeutically active peptides, particularly those requiring asecondary structure to be functional, or PA/PRX302 proteins, toessentially generate an albumin molecule with specific biologicalactivity.

Loops in human albumin structure into which peptides or polypeptides maybe inserted to generate albumin fusion proteins of the inventioninclude: Val54-Asn61, Thr76-Asp89, Ala92-Glu100, Gln170-Ala176,His247-Glu252, Glu266-Glu277, Glu280-His288, Ala362-Glu368,Lys439-Pro447, Val462-Lys475, Thr478-Pro486, and Lys50G-Thr566. Inspecific embodiments, peptides or polypeptides are inserted into theVal54-Asn61, Gln170-Ala176, and/or Lys560-Thr566 loops of mature humanalbumin (SEQ ID NO:27).

IV. Treatment of Prostate Cancer Using Modified Proaerolysin FusionProteins

The variant PA fusion proteins comprising variant PA and albumindisclosed and discussed above are specifically activated to potentcytotoxins within prostate cancer sites via the proteolytic activity ofprostate-specific proteases such as PSA, PSMA, and HK2. Targeting insome examples is achieved by including one or more prostate-tissuespecific binding domains, such as LHRH peptide which can bind to itscognate LHRH receptor expressed by prostate cancer cells, or PSMA orLHRH antibodies, which can bind to PSMA or LHRH expressed on the surfaceof prostate cancer cells. One skilled in the art will recognize that theuse of an albumin-variant PA fusion protein which includes a furincleavage site and an LHRH peptide or antibody, can be used to treatother cancers which express LHRH receptors, such as melanoma and cancersof the breast, ovary and lung, using the albumin-variant PA fusionproteins and methods disclosed herein. Furthermore, one skilled in theart will recognize that the use of an albumin-variant PA fusion proteinwhich includes a furin or PSMA cleavage site, and/or a PSMA antibody,can be used to treat other cancers in which PSMA is expressed (e.g. inthe vasculature of the tumor), such as cancers of the breast, colon,kidney, bladder and brain, using the variant albumin-variant PA fusionproteins and methods disclosed herein.

The disclosed albumin-variant PA fusion proteins, such as nucleic acidsand/or proteins, can be administered systemically or locally using anymethod known in the art, to subjects having localized or metastaticprostate cancer. In addition, the disclosed albumin-variant PA fusionproteins can be administered to a subject for immunostimulatory therapy.Due to the specificity of binding and activation of the disclosedalbumin-variant PA fusion proteins, local and systemic administrationshould have minimal effect on a patient's normal tissues and ideallyproduce little to no side effects.

In one example, the disclosed albumin-variant PA fusion proteins areinjected into the prostate gland (intraprostatically) and/or into theprostate tumor (intratumorally) in a subject having prostate cancer,such as a localized tumor. Such localized injection and subsequent lysisof prostate cancer cells within the prostate gland can produce animmunostimulatory effect leading to a decrease or elimination ofmicrometastatic disease in treated subjects. In this way, systemicdisease is treated or reduced through a minimally toxic, locally appliedtherapy.

In addition, or alternatively, the disclosed albumin-variant PA fusionproteins can be administered systemically, for example intravenously,intramuscularly, subcutaneously, or orally, to a subject having prostatecancer, such as a metastatic prostate tumor. Systemic therapy can alsohave an immunostimulatory anti-tumor effect. The disclosedalbumin-variant PA fusion proteins which include a PSA-cleavage site arenot hydrolyzed by serum proteases or enzymatically inactive PSA withinthe blood. Instead, the unhydrolyzed disclosed variant PA fusionproteins are delivered via the blood to the extracellular fluid withinmetastatic cancer deposits where they can be hydrolyzed to the activetherapeutic toxin by the enzymatically active PSA secreted by theseprostate cancer cells. Once hydrolyzed, the liberated toxin entersPSA-producing and non-producing bystander cells in the immediatevicinity due to its high membrane penetrating ability and induces thecytolytic death of these cells.

An additional method for systemically treating prostate cancer in asubject is also disclosed. In this method, prostate cancer cells areremoved from the subject having prostate cancer, such as a metastaticprostate tumor. Alternatively or in addition, established prostatecancer cell lines can be used. Examples of prostate cancer cell linesthat can be used include, but are not limited to: PSA-producing cellssuch as LNCaP (such as ATTC Nos. CRL-1740 and CRL-10995) and CWR22R(ATCC No. CRL-2505 and Nagabhushan et al., Cancer Res. 56(13):3042-6,1996), or PSA non-producing cells such as PC-3 (ATCC No. CRL-1435) andDU 145 (ATCC No. HTB-81). The removed cells or cell lines are incubatedor contacted with the disclosed albumin-variant PA fusion proteins. Thisincubation results in lysis of the cells by the albumin-variant PAfusion proteins, and production of a cell lysate which is administeredto the subject. In one example, the method further includesadministration of immunostimulatory factors, lysates from prostatecancer cells engineered to produce immunostimulatory factors, and/orirradiated prostate cancer cells (including prostate cancer cellsengineered to produce immunostimulatory factors). Examples ofimmunostimulatory factors include, but are not limited to: granulocytemacrophage colony stimulatory factor (GM-CSF); members of theinterleukin family of proteins such as but not limited to interleukin-2and interleukin-6, granulocyte colony stimulatory factor (G-CSF); andmembers of interferon family such as interferon alpha, beta or gamma.Administration of such materials to a subject can be simultaneous withthe cell lysate (co-administration), before administration of the celllysate, and/or subsequent to administration of the cell lysate.

In one example, such administration enhances the ability of a subject todecrease the volume of a prostate tumor and/or a metastatic tumor. Forexample, the disclosed methods can reduce prostate tumor cell volumeand/or a metastatic tumor cell volume, such as by at least 10%, at least20%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 80%, at least 85%, at least 90%, at least 95% or more. Inaddition, the disclosed methods can result in a decrease in the symptomsassociated with a prostate tumor and/or a metastatic prostate tumor.

The disclosed albumin-variant PA fusion proteins can be administered asa single modality therapy or used in combination with other therapies,such as radiation therapy and/or androgen ablative therapies (such asLHRH receptor agonists/antagonists, antiandrogens, estrogens, adrenalsteroid synthesis inhibitors ketoconazole and aminoglutethimide). Inaddition, administration of the disclosed albumin-variant PA fusionproteins can be alone, or in combination with a pharmaceuticallyacceptable carrier, and/or in combination with other therapeuticcompounds, such as those that reduce the production of antibodies to theadministered variant PA proteins (for example Rituximab and steroids)and other anti-tumor agents.

Without further elaboration, it is believed that one skilled in the art,using the preceding description, can utilize the present invention tothe fullest extent. The following examples are illustrative only, andnot limiting of the remainder of the disclosure in any way whatsoever.

V. Other Proteases and Macromolecules

The present invention also provides proaerolysin fusion proteins thatare activated by proteases other than PSA and/or conjugated tomacromolecules other than HSA. Examples of proteases that can be used ina fusion protein include, but are not limited to, caspase 3, cathepsinB, carboxypeptidase, fibroblast activation protein, MMP-2/-9/-14, MMP7,plasmin, thimet oligopeptiase, uPA, cathepsin K, thrombin, and trypsin.The cleavage site sequences of such proteases are known in the art.Other macromolecules that can be used in place of HSA include, but arenot limited to, polyethylene glycol (PEG), N-(2-hydroxypropyl)methacrylamide copolymer (HPMAcp), and dextran.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices, and/or methods described andclaimed herein are made and evaluated, and are intended to be purelyillustrative and are not intended to limit the scope of what theinventors regard as their invention. Efforts have been made to ensureaccuracy with respect to numbers (e.g., amounts, temperature, etc.) butsome errors and deviations should be accounted for herein. Unlessindicated otherwise, parts are parts by weight, temperature is indegrees Celsius or is at ambient temperature, and pressure is at or nearatmospheric. There are numerous variations and combinations of reactionconditions, e.g., component concentrations, desired solvents, solventmixtures, temperatures, pressures and other reaction ranges andconditions that can be used to optimize the product purity and yieldobtained from the described process. Only reasonable and routineexperimentation will be required to optimize such process conditions.

Background

To overcome tumor cell heterogeneity based therapeutic resistance ofprostate cancer, a strategy was developed by the Denmeade/Isaacs labs tosynthesize “molecular grenades” that are designed to efficiently“detonate” (i.e., release a highly potent killing entity) within theextracellular fluid only at sites of prostate cancer (1). The chemicalengineering requirements for this strategy are that: 1) the killingentity be a highly potent inhibitor of such an essential intracellularprocess that this inhibition kills all cell types without thedevelopment of resistance, 2) this killing entity be capable of couplingvia a peptide bond to a specifically engineered linker peptide producingan initially inactive “molecular grenade”, and 3) the sequence of thislinker peptide is designed so that its efficient hydrolysis and thusliberation of the killing entity is restricted to active enzymes presentonly in the extracellular fluid at sites of metastatic prostate cancer.Thus, when such a chemically engineered “molecular grenade” is infused,it distributes systemically throughout the body but can only beactivated (i.e., detonated) by a protease expressed within theextracellular fluid (ECF) within metastatic sites of prostate cancer,but not within sites of non-prostate normal tissue. Within tumor sites,the protease “pulls the pin” on the grenade by proteolytically releasingthe highly potent killing entity.

The advantage of such selective extracellular hydrolysis is that only afraction of the cancer cells need to express the enzyme since itscontinuous activity amplifies the level of the killing toxin liberatedwithin the ECF shared by all cells within the metastatic site. Thisamplification minimizes the problem of tumor cell heterogeneity byinducing a substantial “bystander effect” in which, like a detonatedgrenade, all cells within the tumor site including both malignant andinfiltrating host supportive cells are killed, even those that do notexpress the activating enzyme. Thus, development of resistance isretarded without simultaneously producing non-selective host toxicity.

For this application, we are focused upon using as the killing entitythe bacterial toxin proaerolysin (PA). PA is produced and secreted bythe aquatic Gram-negative bacteria Aeromonas hydrophilia and is ideallysuited for such recombinant modification. These bacteria synthesize the53-kDa PA protein and secrete it as a water soluble dimer. PA contains anumber of separate domains that include an N-terminal GPI-anchoredprotein binding domain, a “toxin” domain responsible for pore formation,a protease activation domain and a C-terminal inhibitory peptide thatrenders the toxin inactive until it is proteolytically released (2).

The natural protease activation domain is a six amino acid sequence thatis a proteolytic substrate for ubiquitous furin-like proteases producedby many cell types. Hydrolysis of the activation domain by furinliberates the C-terminal inhibitory peptide converting inactive PA intocytotoxic aerolysin. Activated aerolysin binds to GPI-anchored proteinsin the cell membrane and inserts into the membrane forming a highlystable ˜17 Å heptamer channel, which leads to rapid cell swelling andresultant osmotic cell death which is proliferation independent andwithout a mechanism for the development of resistance (2).

Most mammalian cells, with the exception of erythrocytes, produce thenecessary furin protease required to activate proaerolysin (2). Onceactivated, wild-type aerolysin is toxic to mammalian cells, includingerythrocytes, at picomolar concentrations. In our laboratory, wild-typePA is toxic to all human prostate cancer cell lines (i.e., PC-3, DU145,LNCaP, LAPC-4, CWR22Rv1) and human non-prostate cancer cell lines [i.e.,TSU (bladder origin) SN12C (renal cell), TT (medullary thyroid)] withLD50 values of 50 picomolar or less (2).

As expected from its mechanism of cytotoxicity, PA is very toxic invivo. Following a single intravenous dose, the LD100 (i.e., the dosethat kills 100% of animals within 24 hrs) in mice, is 0.1 μg (2). Thus,PA must be modified to target its killing ability to sites of prostatecancer without producing unacceptable host toxicity. An enablingprinciple for such a “molecular grenade” approach is that only normaland malignant prostate epithelial cells synthesize and secrete highamounts of enzymatically active Prostate Specific Antigen (PSA) intotheir ECF (3). Once in the ECF, enzymatically active PSA eventuallyenters the blood where it is inactivated by binding to major serumprotease inhibitors [i.e., a1-antichymotrypsin and a2-macroglobulin] forPSA (3). Therefore, the only location in the body besides the prostatein which PSA is enzymatically active is in the ECF within sites ofprostate cancers (3).

Previously, we identified a specific 7 amino-acid peptide sequence(i.e., HSSKLQ, SEQ ID NO:2) that is efficiently and selectively cleavedafter Q by PSA (3). To allow selective PSA proteolysis and thusactivation, the present inventors genetically engineered a bacteriallyproduced recombinant PA in which the furin cleavage sequence wassubjected to site-directed mutagenesis using polymerase chain reactionto convert it to a PSA-cleavable sequence [HSSKLQ] (2). The mutated genewas then subcloned into the pMMB66HE vector for amplification inEscherichia coli. This construct was then transferred to aprotease-deficient strain of A. salmonicida that facilitates theproduction of large amounts of uncontaminated PRX302. This modifiedtoxin, termed PRX-302 is activated in culture by PSA producing prostatecancer cells resulting in potent cell killing (i.e., LD50 values of lessthan 200 picoMolar, but only against PSA producing cancer lines) (2).When as little as 100 picomoles of PXR302 is injected directly into PSAexpressing human prostate cancer xenografts in nude mice, sufficienthydrolysis of the protoxin occurs in the extracellular fluid viaenzymatically active PSA to produce more than a 50% regression of thecancer in all animals by 2 weeks post injection with 25% of the injectedanimal having a complete regression by a month post treatment (2). Incontrast, no tumor regression is produced if PRX302 is injectedintra-tumorally into non-PSA producing non-prostate cancers (2).

While PRX302 is highly water-soluble, PSA-activation releases theC-terminal inhibitory peptide causing a conformational change in theprotein that exposes hydrophobic domains that result in rapid membraneinsertion. Therefore, once activated in the extracellular fluid at sitesof prostate cancer, very little of the aerolysin toxin leaks back intothe systemic circulation limiting the non-specific toxicity to hosttissue. This was documented by preclinical studies demonstrating thatintraprostatic injection into the PSA-producing monkey prostate producedno toxicity in periprostatic tissues, including the lateral pelvicfascia, anal sphincter, urethra, urinary bladder, or rectum or otherdistant organs (2).

Based on efficacy and safety in these preclinical studies, PRX302 hasundergone an open-label phase 1 dose-escalation trial to assess thesafety of transperineal injection of PRX302 as therapy for localradiation-recurrent prostate cancer or BPH (4). Subsequently, a phase 2volume-escalation study was performed to evaluate the effect of PRX302on alleviating lower urinary tract systems in men with moderate tosevere BPH. The results of these phase II trial have been reportedrecently and document the safety and therapeutic activity of a singletransperineal, intraprostatic treatment of PRX302 over a follow-upperiod of a year (4).

Presently, more than 120 patients have received PRX302 which is enteringphase III registration trials as local therapy for symptomatic BPH.Based upon these encouraging clinical results for local delivery ofPRX302, the possibility that a therapeutic effect is produced when thePRX302 is administered systemically was tested. Because the modifiedtoxin is injected intravenously when administered for systemic treatmentof metastatic prostate cancer, we determined whether PRX302 is stable tonon-specific activation in normal and PSA-containing human plasmautilizing a sensitive hemolysis assay. These studies documented thatpre-incubation of the PRX302 with enzymatically active PSA in aqueousbuffer alone prior to adding RBC's results in ˜45% hemolysis. To assesswhether PRX302 becomes activated in either normal human plasma or plasmafrom prostate cancer patients with elevated PSA, PRX302 (50 ng/ml) wasadded to both 50% unmodified human plasma and 50% plasma pre-incubatedwith 10,000 ng/ml of enzymatically active PSA in order for the PSA toform enzymatically inactive complexes with serum protease inhibitors,and then the plasma was incubated with human red blood cells (2% v/v).The addition of PRX302 to human plasma or human plasma spiked with highconcentration of PSA results in no appreciable hemolysis (i.e., <1% ofTriton control). These results demonstrate that the PRX302 can beadministered systemically without any significant activation in theblood even when it contains an extremely high level (i.e., 10,000 ng/ml)of measurable but inactivated PSA.

Next, we determined the maximally tolerated systemic dose of wild typePA vs. PRX302 when given IV to nude mice. The wild type PA is highlytoxic to mice. An intravenous dose of 1 μg causes death within one hourand the LD100 at 24 hrs following a single IV injection is 0.1 μg. Incontrast, the LD100 of a single IV injection of the PRX302 at 24 hrspost injection was found to be 25-fold higher (i.e., 2.5 μg total dose).Additional experiments, however, demonstrated that mice could only besafely injected with 1 μg daily of PRX302 for 5 consecutive days withoutsignificant toxicity. Higher doses resulted in animal deaths ˜1 weekafter multiplied daily doses >1 μg. Preliminary studies are consistentwith the dose limiting in vivo toxicity of the wild type and modifiedPRX302 being due to its binding to GPI-anchor proteins expressed by mostmammalian cell types. Nude mice bearing the LNCaP human prostate cancerwere given the maximally tolerated dose regimen of 1 μg/day of PRX302given IV for 5 days. In this experiment there was no significantdifference in LNCaP tumor size out to 20 days post-therapy in the PRX302treated group vs. controls.

While PRX302 is effective as local therapy for prostate cancer, theselatter results illustrate that it is not possible to give sufficientPRX302 systemically to achieve an effective level within sites ofprostate cancer without producing unacceptable host toxicity due to itsbinding to GPI-anchor proteins ubiquitously expressed on cells in thehost normal tissue.

Experimental Design

Previous studies document that the binding to the GPI-anchor proteininvolves a surface composed of regions of domain 1 (i.e., which includesthe N-terminus) and domain 2 of PA and that when a bulky protein isfused to the N-terminal, such binding is prevented (5). These resultsraise the possibility that by fusing a bulky protein to the N-terminusof PRX302, binding to the GPI-anchor can be prevented thus allowing thevariant PRX302 protein to be delivered systemically without non-specificGPI-dependent uptake by normal tissues. This raises the questions ofboth what “bulky protein” and how should it be coupled in such arecombinant variant protein so that the “bulky protein” can behydrolyzed off to selectively liberate PRX302 with its GPI-anchorbinding surface free to bind to GPI-anchors only on cells in sites ofmetastatic prostate cancer.

Along these lines, human serum albumin (HSA) is an ideal candidate. HSAis the most abundant plasma protein (35-50 g/L serum protein) and has amolecular weight of 67 kDa and serum half-life of 20 days. Tumors trapplasma proteins and utilize their degradation products for proliferation(6). Pharmacokinetic profiling documented that such tumor uptake iscorrelated with both serum half-life and protein weight. Such tumoruptake, tumor blood flow and the transport of molecules in theinterstitium led Maeda and Matsumura to coin the expression “EPR” (i.e.,enhanced permeability and retention of macromolecules) in relation topassive tumor targeting of serum proteins. The leaky defective bloodvessels of tumor tissue make its vasculature permeable formacromolecules whereas in blood vessels of healthy tissue only smallmolecules can pass the endothelial barrier. The pore size of tumormicrovessels varies from 100 to 1200 nm in diameter and serum albuminhas an effective diameter of 7.2 nm allowing extravasation into tumortissue but not into normal tissue (6).

Thus, the hypothesis for this application is that the C-terminus of HSAcan be fused via a PSA cleavable peptide linker (i.e., HSSKLQ, SEQ IDNO:2) to the N-terminus of PRX302 to generate a novel recombinant PRX302protein which will not bind to GPI-anchored proteins on normal cells inthe blood or host tissues. Instead, it will accumulate via an EPR effectwithin sites of metastatic prostate cancer where enzymatically activePSA in the extracellular fluid will hydrolyze the HSA linker liberatingPRX302. Additionally, PSA will also remove the C-terminal inhibitorypeptide from PRX302 to generate aerolysin monomers which can oligomerizeto form the heptameric pores that will result in selective killing ofcells only in the site of metastatic prostate cancer.

REFERENCES

1. Denmeade S R, Isaacs J T. Engineering enzymatically activated“molecular grenades” for cancer. Oncotarget 2012; 3: 666-7.

2. Williams S A, Merchant R F, Garrett-Mayer E, Isaacs J T, Buckley J T,Denmeade S R. A prostate-specific antigen-activated channel-formingtoxin as therapy for prostatic disease. J Natl Cancer Inst. 2007; 99:376-85.

3. Denmeade S R, Sokoll L J, Chan D W, Khan S R, Isaacs J T.Concentration of enzymatically active prostate-specific antigen (PSA) inthe extracellular fluid of primary human prostate cancers and humanprostatic cancer xenograft models. Prostate 2001; 48:1-6.

4. Denmeade S R, Egerdie B, Steinhoff, et al. Phase 1 and 2 studiesdemonstrate the safety of intraprostatic injection of PXR302 for thetargeted treatment of lower urinary tract symptoms secondary to benignprostatic hyperplasia. Eur Urol 2011; 59: 747-54.

5. Osusky M, Teschke L, Wang X et al. A chimera of interleukin 2 bindingvariant of aerolysin is selectively toxic to cells displaying theinterleukin 2 receptor. J Biol Chem 2008; 283: 1572-1579.

6. Kratz F. Albumin as a drug carrier: design of prodrugs, drugconjugates, and nanoparticles. J Controlled Release 2008; 132: 171-183.

7. Denmeade S R, Mhaka A M, Rosen D M, Brennen W N, Dalrymple S P, DachI, Olesen Claus, Gurel B, DeMarzo A M, Wilding G, Carducci M A, Dionne CA, Møller J V, Nissen P, Christensen S B, Isaacs J T. Engineering aProstate-specific Membrane Antigen-Activated Tumor Endothelial CellProdrug for Cancer Therapy. Sci Trans Med 2012; 4: 140ra86.

We claim:
 1. A recombinant protein comprising SEQ ID NO:48.
 2. Therecombinant protein of claim 1, wherein the protein further comprises apolyhistidine tag.
 3. The recombinant protein of claim 2, wherein thepolyhistidine tag comprises six histidines at the C-terminus of SEQ IDNO:48.